Improving The Efficiency of NADPH-dependent Xylitol Production in Engineered Escherichia coli

Open Access
Author:
Akinterinwa, Olubolaji
Graduate Program:
Chemical Engineering
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
July 23, 2010
Committee Members:
  • Andrew Michael Cirino, Dissertation Advisor
  • Patrick C Cirino, Committee Chair
  • Donald Ashley Bryant, Committee Member
  • Costas D Maranas, Committee Member
  • Andrew Zydney, Committee Member
Keywords:
  • biocatalysis
  • Escherichia coli
  • metabolic engineering
  • xylitol
  • NADPH
Abstract:
The development of cost-effective and sustainable microbial processes for the production of chemicals and high-energy fuels is crucial to reducing current dependence on petroleum. Nature has provided a vast collection of biological systems that can be redesigned to efficiently convert readily available, inexpensive, biomass-derived sugars to these high-value products. In this study we describe various metabolic engineering strategies aimed towards developing the E. coli bacterium as a microbial host for heterologous, nicotinamide cofactor (NADH and NADPH)-dependent transformations. These transformations are of major importance because they are applicable in the synthesis of a variety of important chemicals. NADPH-dependent xylitol production from glucose/xylose mixtures serves as the experimental platform and is used to illustrate the important aspects of this work. The experimental system is designed such that glucose oxidation serves as the source of electrons for xylose reduction to xylitol. Previous assessments of the metabolic behavior of this system under aerobic conditions showed that xylose conversion to xylitol was less than 100% due to production of xylulose as a by-product. It was also shown that cofactors were not efficiently diverted towards xylitol production and this resulted in sub-optimal yields (defined as mol of xylitol produced/ glucose consumed and denoted as YRPG) under these conditions. Therefore in this study genetic modifications and process conditions aimed at improving xylose conversion to xylitol, the efficiency of the biotransformation (defined as experimental YRPG / theoretical YRPG) and the productivity of the engineered system are investigated. We focus on parameters such as competing pathways, transhydrogenase activity and level of aeration, as these are key factors expected to impact cofactor availability for the desired biotransformations. We find that efficiency of the biotransformation is maximal under non-respiratory conditions possibly due to the elimination of respiration as a sink of reduced cofactors.